A sensitive and specific enzyme‐linked immunosorbent assay for the detection of lincomycin in food samples

BACKGROUND: Lincomycin (LIN) is an antibiotic widely used in veterinary medicine to cure infections caused by Gram‐positive pathogens. Although the toxicity of LIN is not serious, it will cause adverse effects in humans, such as pseudomembranous enteritis and bacterial resistance. In this study, for the preparation of a LIN derivative, a novel modification method was adopted. The LIN derivative modified at 2‐position with a carboxylic group at the end of the spacer was synthesised and coupled to carrier proteins. A LIN polyclonal antibody‐based enzyme‐linked immunosorbent assay (ELISA) was developed and characterised. RESULTS: The ELISA standard curve was constructed with concentrations of 0.1–1000 ng mL^?1. The IC₅₀ value for nine standard curves was in the range 23.7–29.3 ng mL^?1 and the limit of detection at a signal‐to‐noise ratio of 3 was 0.15–0.98 ng mL^?1. The cross‐reactivity value of the LIN antibody with clindamycin hydrochloride, a homologue of LIN with similar molecular structure, was 18.9%, while less than 0.1% cross‐reactivity was found with seven other compounds. For LIN‐spiked food samples, the recoveries and relative standard deviation (RSD) were 76.6–117.6% and 1.7–34.6%, respectively. CONCLUSION: The proposed ELISA can be utilised as a sensitive and specific analytical tool for the detection of LIN in food samples. Copyright © 2010 Society of Chemical Industry     

磁分散固相萃取-酶联免疫吸附法检测金黄色葡萄球菌肠毒素A

目的 以金黄色葡萄球菌肠毒素A (staphylococcal enterotoxin A, SEA)为代表,设计并合成一种适配体修饰功能化磁性材料,与酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)检测技术相结合,进一步提高检测方法灵敏度。方法 样品经SEA适配体修饰磁性微球富集后,结合SEA的ELISA试剂盒检测样品中SEA含量,建立SEA检测的磁分散固相萃取-酶联免疫吸附新方法。结果 与仅采用ELISA试剂盒测定SEA的方法相比,本研究方法的灵敏度可提高10倍,检测方法的线性范围为0.01～0.81μg/L,同时检测方法具有良好的特异性。将建立的检测方法用于牛奶中SEA的检测,回收率在84.45%～114.45%之间。结论 本方法操作简单、特异性强、灵敏度高,同时具有广泛的适用性,可通过特定适配体修饰磁性微球实现不同目标物的检测,同时适配体修饰功能化磁性材料也可与其他检测技术联合使用,提高检测的灵敏度。     

双抗夹心酶联免疫吸附法检测荞麦过敏蛋白

提取制备荞麦过敏原蛋白（TBt）并免疫动物,分别制备鼠多克隆抗体和兔多克隆抗体,建立了双抗夹心酶联免疫吸附检测法（ELISA）用于检测食品中的荞麦过敏原。SDS-PAGE结果表明,纯化的TBt纯度达到98%以上,鼠抗血清效价为1∶6400。建立的双抗夹心ELISA法对荞麦过敏原蛋白的最低检测限为0.16μg/m L,线性范围为0.16¹6μg/m L。并采用建立的双抗夹心ELISA法对市场出售的15种食品中荞麦过敏成分进行了检测。结果显示,该方法对绝大多数食品中的荞麦过敏原都有很好的特异性及灵敏性,说明该法可用于食物过敏原的诊断及食品中微量荞麦过敏成分的检测。      

竞争抑制酶联免疫吸附法快速检测金黄色葡萄球菌肠毒素P

目的 建立一种稳定性强、灵敏度高的快速检测金黄色葡萄球菌新型肠毒素P(staphylococcal enterotoxin P,SEP)的单克隆抗体竞争抑制酶联免疫吸附法(Enzyme-linked Immunosorbent assay,ELISA)。方法 根据ELISA的检测程序,利用交叉方阵滴定法确定SEP抗原的最佳包被浓度及单克隆抗体的最佳倍比稀释浓度,再对辣根过氧化物酶标记的羊抗鼠IgG(HRP-IgG)最佳稀释浓度及最佳反应时间进行筛选,然后通过测定450nm处不同SEP抗原包被条件(包被液类型、包被环境)、封闭液类型及浓度、封闭时间、竞争反应温度、反应方式(预混反应、直接反应)下的OD值对检测条件进行优化,最后用灵敏度、批内变异、批间变异和加标样品回收率对方法进行评价。结果 SEP抗原最佳包被浓度为2.5 μg/mL,鼠单抗血清稀释度1:6000,酶标抗稀释度1:3000;反应1 h,最佳包被条件为磷酸盐缓冲液4 ℃过夜,10%脱脂乳封闭2 h,37 ℃直接竞争反应。此方法的线性回归方程为：y = 0.4166x - 0.7415(R2 = 0.9908),灵敏度为0.954 μg/mL,批内及批间变异系数均低于3 %,对人工污染的脱脂牛奶、LB液体培养基中肠毒素蛋白SEP的回收率高于98%。结论 本实验建立了一种能够快速检测葡萄球菌肠毒素SEP的ELISA方法。     

Development of antipeptide enzyme‐linked immunosorbent assay for determination of gelatin in confectionery products

The gelatin sources have become a controversial issue with regard to religious and health concern. Thus, the aims of this study were to develop and evaluate the efficiency of polyclonal antibodies against peptide immunogen of collagen α2 (I) chain for determination of gelatin sources in confectionery products by competitive indirect enzyme‐linked immunosorbent assay (ELISA). Collagen α2 (I) chain protein showed resistance against heat treatment and detectable in certain commercial products when analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE). The established ELISA exhibited low cross‐reactivity to fish and chicken gelatin. The IC₅₀ value was 0.39 μg mL^?1, and the limit of detection (IC₁₀) was 0.05 μg mL^?1. There were no false‐positive results from forty‐eight commercially processed products. The present method is useful for determination of gelatin in confectionery products.     

酶联免疫吸附法快速检测饲料中赭曲霉毒素A

目的建立一种酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)快速检测饲料中赭曲霉毒素A快速的方法。方法样品采用60%的甲醇溶液提取,提取液经过离心、快速滤纸过滤后,用稀释缓冲溶液按10倍的比例对提取液进行稀释即为试样液,采用酶联免疫吸附方法进行检测。结果在加标浓度为25、50和100μg/kg的加标水平下,赭曲霉毒素A在饲料中的加标回收率为88.4%~134.6%,变异系数为3.5%~6.9%。抽取10份样品进行检测,赫曲霉毒素A含量在14.1~38.2μg/kg。符合国家的标准GB 13078-2017对饲料中赫曲霉毒素A含量的规定。结论本方法灵敏度高、快速简便,适用于饲料中赭曲霉毒素A的快速批量检测。     

一种喹诺酮类药物的酶联免疫快速检测试剂盒的研制

利用酶联免疫技术研制了一种快速检测鱼肉中的喹诺酮类药物试剂盒,经过测试,该试剂盒对鱼肉样本中氧氟沙星、环丙沙星、氨氟沙星等3种喹诺酮类药物的检测限为1μg/kg,批内、批间相对标准偏差均小于10%;稳定性测试结果表明,试剂盒能够在4℃条件下保存12个月,37℃条件下保存7d。喹诺酮类药物单克隆抗体与氧氟沙星、环丙沙星、氨氟沙星的交叉反应率均为100%。该方法准确,可靠,使用简便,适合大量样品的现场检测。     

呋喃它酮代谢物间接竞争酶联免疫检测方法的建立

目的建立呋喃它酮代谢物5-甲基吗啉-3-氨基-2-唑烷基酮(5-morpholine-methyl-3-amino-2-oxazolidinone,AMOZ)间接竞争酶联免疫检测法。方法通过衍生化反应制备了人工抗原CPAMOZ并用活化酯法将其与鸡卵清蛋白(ovalbumin,OVA)连接成为包被原CPAMOZ-OVA。对包被原和单克隆抗体的最佳工作浓度、封闭液浓度、竞争时间、辣根过氧化物酶标记羊抗鼠二抗(horseradish peroxidase labelled goat antimouse Ig G conjugate,HRP-Ig G)、孵育时间和显色反应时间进行优化,并对方法灵敏度、特异性、精密度和准确度进行方法学评价。结果最优条件下,包被原和单克隆抗体稀释倍数分别为500倍和2000倍,封闭液浓度为2%,竞争时间为30 min,二抗孵育时间为30 min,显色时间为15 min。所建立标准曲线的对应线性方程是Y=-0.439X+0.670(r~2=0.992),IC_50为2.439 ng/m L,线性范围为0.506~11.765 ng/m L,回收率为86.7%~90.1%,批内和批间变异系数分别为1.4%~5.8%和2.1%~4.9%。结论该方法特异性强,准确性、精密度和重现性良好,可用于对AMOZ的快速筛查。     

一种叶酸的酶联免疫快速检测试剂盒的研制

利用酶联免疫技术研制一种快速检测食品中的叶酸试剂盒,经过测试,该试剂盒对牛奶样本的检测限为10.0μg/L,批内、批间相对标准偏差均小于10%;稳定性测试结果表明,试剂盒能够在4℃保存12个月,37℃保存7d。叶酸单克隆抗体与二氢叶酸、四氢叶酸的交叉反应率均小于4%。该方法准确,可靠,使用简便,适合大量样品的现场检测。      

西马特罗单克隆抗体制备以及酶联免疫试剂盒的研究

通过制备西马特罗半抗原,载体蛋白与之偶联合成免疫原,免疫原用于免疫动物,获得抗西马特罗单克隆抗体,制备能够检测猪尿中西马特罗残留量的酶联免疫试剂盒。结果表明,该试剂盒对猪尿中西马特罗的检测限为0.295 μg/L,半抑制浓度（IC50）值为0.870 μg/L,回收率范围为81.5%～103.5%,标准曲线线性范围为0.1～8.1 μg/L,精密度试验结果批内、批间的相对标准偏差（RSD）均＜15%,表明该检测方法准确度和精密度高。该试剂盒特异性较好,于4 ℃条件下能够保存12个月,稳定性较好,可以用于猪尿中西马特罗德快速检测。     

双抗体夹心酶联免疫吸附法检测鱼糜制品中鸡蛋卵白蛋白

目的 建立双抗体夹心酶联免疫吸附(sandwich enzyme linked immunosorbent assay, sELISA)法检测鸡蛋卵白蛋白(ovalbumin, OVA)的方法,组装定量检测鱼糜制品中OVA含量的ELISA检测试剂盒。方法 确定各步骤反应时间,使用棋盘法确定捕获抗体和检测抗体的最佳工作浓度,建立检测方法并对其性能进行评价。结果 反应最佳条件为:抗原孵育20 min,检测抗体孵育20min,酶促反应显色10 min。兔抗OVA抗体为捕获抗体,工作质量浓度为1μg/mL,辣根过氧化物酶(horseradish peroxidase, HRP)标记兔抗OVA抗体为检测抗体, 1:5000 (V:V)稀释。采用四参数逻辑曲线拟合标准曲线,所建立sELISA方法的检测范围为8.5～200.0 ng/mL,检出限为3.5 ng/mL,定量限为8.5 ng/mL。批次内和批次间的变异系数分别为2.31%～4.58%和6.06%～12.23%;鱼糜基质中的回收率为80%～130%;测得28种常见食物样品中4种样品的交叉反应率小于0.002%;试剂盒在4℃保存6个月期间,...     

草甘膦单克隆抗体的制备及酶联免疫分析方法的建立

目的 制备出草甘膦(glyphosate,GLY)单克隆抗体,建立间接竞争酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)快速检测茶叶中GLY的方法。方法 首先合成GLY完全抗原(包被原和免疫原),通过免疫小鼠成功制备出GLY单克隆抗体。根据ELISA的检测流程,采用棋盘法确定最佳包被抗原和抗体的稀释倍数,确定最佳包被的温度和时间,并确定最佳加入一抗、二抗后反应时间,建立检测方法并对其性能进行评价。结果 GLY包被抗原和抗体的稀释倍数为1:2000,包被温度为37℃、包被时间为90 min,加入一抗、二抗后反应时间为60min。该方法的线性方程为Y=-0.2353X+0.6539(r²=0.9871),半抑制浓度(50%inhibiting concentration, IC₅₀)为4.508 ng/mL,检出限为1.18 ng/mL。变异系数均在10%以下,与异菌脲、多菌灵、三唑磷、甲基对硫磷、噻菌灵这5种标准品的交叉反应率均低于0.03%,在茶叶中加标回收率为90.86%～110.35%,...     

制备抗五氯酚钠单克隆抗体及其应用于酶联免疫试剂盒的初步研究

本文通过五氯酚钠与4-溴丁酸乙酯经过一系列反应,得到半抗原,制备出抗五氯酚钠单克隆抗体,然后利用酶联免疫技术研制了一种快速检测鱼肉、虾肉中的五氯酚钠试剂盒,经过测试,该试剂盒对鱼肉、虾肉样本的检测限为0.75μg/kg,IC50为0.31μg/L,平均回收率为71.5%¹04.7%,试剂盒的标准曲线范围为0.05⁴.05μg/L,批内、批间相对标准偏差小于10%,稳定性良好。      

酶联免疫法检测酱油中的黄曲霉毒素B1

目的建立酶联免疫法检测酱油中黄曲霉毒素B_1(Aflatoxin B_1,AFB_1)的分析方法。方法酱油样本经纯乙腈(料液比=1:2,V:V)提取,再做1:9稀释,通过酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)测定样本中AFB_1。结果当加标浓度为2μg/kg和5μg/kg时,纯乙腈对酱油中AFB_1的提取回收率结果分别为121.3%和106.5%;方法检出限为1μg/kg。与国标方法检测结果的相对标准偏差小于10%。结论本方法准确、灵敏度高,可适用于酱油中AFB_1的检测。     

酶联免疫吸附法在食品安全性指标检测中的 研究进展

随着我国社会经济的快速发展和人们生活水平的提高,食品安全问题日益成为人们关注的焦点。酶联免疫吸附法是在免疫学和细胞工程学基础上发展的一种微量检测技术,具有操作简便、灵敏度高等优点。本文总结了酶联免疫吸附法在农药残留、兽药残留、致病微生物、生物毒素、非法添加的非食用物质、重金属、过敏原和转基因食品等检测中的研究进展,评价了该检测方法的优缺点,并对其未来的发展方向进行了展望和建议。     

动物组织中呋喃妥因代谢物残留酶联免疫试剂盒的研制

通过对呋喃妥因代谢物分子结构进行改造,制备了半抗原及人工抗原,免疫动物,制备特异性强的单克隆抗体。在筛选呋喃妥因代谢物单克隆抗体的基础上,建立酶联免疫检测方法,并研制出检测动物组织中呋喃妥因代谢物残留的试剂盒。该试剂盒的IC50值为0.094μg/L,最低检测限为0.1μg/kg,样本添加回收率为78.2%～102.7%,变异系数为5.1%～11.4%;与呋喃妥因的交叉反应率为13%,与其他药物的交叉反应率均小于1%;对实际样本的检测结果与液相色谱-串联质谱法基本一致。该方法快速、灵敏、可靠,为动物组织中呋喃妥因代谢物的残留检测提供了便捷、准确的分析检测手段。      

Highly sensitive and specific detection of neonicotinoid insecticide imidacloprid in environmental and food samples by a polyclonal antibody-based enzyme-linked immunosorbent assay

BACKGROUND: Imidacloprid is one of the main neonicotinoid insecticides widely used in agriculture owing its broad spectrum of activity and low bioaccumulation. However, imidacloprid is toxic to honey bees and other beneficial organisms, and its residues may occur in environmental and food samples, posing a potential hazard to consumers. In this study the imidacloprid derivative bearing a three‐atom length spacer was synthesized and coupled to carrier proteins. Highly sensitive and specific polyclonal antibodies against imidacloprid were successfully produced and the polyclonal antibody‐based enzyme‐linked immunosorbent assay (pAb‐ELISA) was developed. RESULTS: The ELISA standard curve was constructed within the concentration range 0.1–100 ng mL^?1. The IC₅₀ value for nine standard curves was in the range 1.2–3.0 ng mL^?1 and the limit of detection was 0.03–0.16 ng mL^?1. The sensitivity of the assay was one order of magnitude higher than that in most published papers. There was almost no cross‐reactivity of the antibody with four structurally related compounds (acetamiprid, nicotine, clothianidin and nitenpyram) and six other compounds, indicating that the assay displays not only high sensitivity but also high specificity. No detectable imidacloprid was found in 11 collected environmental and food samples by the assay. For imidacloprid‐spiked samples, acceptable recoveries of 73.4–94.4% and intra‐assay coefficients of variation of 2.2–12.8% were obtained. The assay was also validated with high‐performance liquid chromatography (HPLC) and a good correlation of ELISA with HPLC was achieved. CONCLUSION: The proposed ELISA provides a sensitive, specific, simple and cost‐effective quantitative/screening method for detecting imidacloprid in environmental and food samples. Copyright © 2011 Society of Chemical Industry     

Quantum dot bead-based competitive immunochromatographic assay for enterotoxin aureus A detection in pasteurized milk

Staphylococcal enterotoxin A (SEA) is an important biotoxin, produced by Staphylococcus aureus under appropriate conditions, and often contaminates milk and dairy products. Herein, an anti-SEA monoclonal antibody (anti-SEA mAb) was prepared by injecting the SEA protein in BALB/c mice, and a novel immunochromatographic assay (ICA) was developed for the rapid and sensitive determination of SEA in pasteurized milk by using highly luminescent quantum dot beads (QB) as signal amplification probe. Given the 1020-fold enhancement in the photoluminescence intensity of QB to the original quantum dot, the proposed QB-ICA exhibits high sensitivity for SEA determination in real milk samples with a limit of detection of 1.89 ng/mL, and shows good dynamic linearity for SEA quantitative detection from 2 to 150 ng/mL within 15 min of test time. The proposed QB-ICA also shows good selectivity to SEA detection with a negligible cross-reaction to common analogs, including staphylococcal enterotoxins B, C, D, and E. In addition, the accuracy and precision of QB-ICA were assessed by analyzing SEA-fortified milk samples. The average recoveries of intra- and interassays range from 85.5 to 128.1%, and the coefficients of variation range from 4.6 to 14.2%, indicating an acceptable accuracy for the quantitative detection of SEA in real milk samples. In summary, this work provides a powerful and rapid analysis tool for the sensitive monitoring of SEA contamination in pasteurized milk samples.